Cytological stain composition

ABSTRACT

The present invention relates to the analysis of cytological material. Specifically, the invention relates to stains and methods of producing the stains, methods of staining cells for cytological or histological analysis to contrast the nuclear portion of the cell from the cytoplasmic portion, and systems and methods for illuminating and imaging a cytological sample. The analysis can be automated or manual.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part and claims the benefitof U.S. patent application Ser. No. 09/430,116, filed Oct. 29, 1999, thedisclosure of which is hereby incorporated herein by reference in itsentirety. In addition, this application incorporates herein by referenceU.S. patent application Ser. Nos. 09/430,117, 09/430,196, and09/430,198, each filed Oct. 29, 1999.

TECHNICAL FIELD

[0002] The present invention relates to the analysis of cytologicalmaterial. Specifically, the invention relates to stains and methods ofproducing the stains, methods of staining cells for cytological orhistological analysis to contrast the nuclear portion of the cell fromthe cytoplasmic portion, and systems and methods for illuminating acytological sample. The analysis can be automated or manual.

BACKGROUND INFORMATION

[0003] Cytology is the branch of biology dealing with the study of theformation, structure, and function of cells. As applied in a laboratorysetting, cytologists, cytotechnologists, and other medical professionalsmake medical diagnoses of a patient's condition based on visualexamination of a specimen of the patient's cells. A typical cytologicaltechnique is a “Pap smear” test, in which cells are scraped from awoman's cervix and analyzed in order to detect the presence of abnormalcells, a precursor to the onset of cervical cancer. Cytologicaltechniques are also used to detect abnormal cells and disease in otherparts of the human body.

[0004] Cytological techniques are widely employed because collection ofcell samples for analysis is generally less invasive than traditionalsurgical pathological procedures such as biopsies, whereby a tissuespecimen is excised from the patient using specialized biopsy needleshaving spring loaded translatable stylets, fixed cannulae, and the like.Cell samples may be obtained from the patient by a variety of techniquesincluding, for example, by scraping or swabbing an area, or by using aneedle to aspirate body fluids from the chest cavity, bladder, spinalcanal, or other appropriate area. The cell samples are placed insolution and subsequently collected and transferred to a glass slide forviewing under magnification. Fixative and staining solutions aretypically applied to the cells on the glass slide, often called a cellsmear, for facilitating examination and for preserving the specimen forarchival purposes.

[0005] A traditional multicolored stain is desirable for staining cellsmears for certain cytological analyses. It is advantageous to stain thenucleus and the cytoplasm of the specimen with different colors, so thatthe nuclear material and cytoplasmic material can be readilydistinguished, either visually or by automated imaging equipment. In onestaining practice, the cytoplasm is transparent, whereas the nucleus istransparent to opaque. This staining pattern allows the cytologist todistinguish cells that are morphologically abnormal indicated, forexample, by nuclear material that is excessively large and/or dark incolor. In addition, cytologists find the variety of colors of thetraditional stains, particularly the Papanicolaou stain, helpful toreduce eyestrain and to aid diagnosis.

[0006] Traditional stains, including the Papanicolaou stain, aredifficult for an automated system to analyze. The variety of colors inthe cytoplasm from traditional stains, which are straightforward for thehuman eye to distinguish, are not readily analyzed with automatedimaging systems, because they contrast to varying degrees with thetraditional blue hematoxylin stain of the nucleus. The varying contrastmakes automated analysis unreliable.

[0007] During the approximately seventy years since its introduction,the original Papanicolaou stain has undergone many modifications.Currently, the dyes, reagents, and methodology vary widely based on thepreferences of each laboratory. While standardization of aPapanicolaou-like stain has been proposed for many years, there has beenlittle incentive for laboratories to do so. This variability affectscurrent imaging technologies which may reject numerous slides eitherbecause of problems inherent with a conventional Pap smear preparation,or because of poor staining that produces nuclear-cytoplasmic contrastthat is inadequate for image acquisition and analysis.

[0008] A number of researchers have developed algorithms in an attemptto attain automated analysis of cells stained with the multicoloredPapanicolaou stain. Such techniques involve the use of variousinstrumental artifacts, such as different colors of light, filters, andcolor television cameras. Many require a high level of sophisticationthat is costly in terms of hardware and software. Further, theseapproaches have not proven accurate and reliable enough to be widelyused in clinical cytological and histological diagnoses.

[0009] Conventional machine vision illumination sources are lowefficiency broadband sources such as tungsten-halogen, sodium-halide, orxenon lamps. These sources convert a small percentage of their inputenergy to broadband light. Accordingly, efficiency drops significantlyin a cytological application that requires a narrow band light source.Typically, these devices generate a significant amount of heat, requirefilters for obtaining correct wavelengths, and are relatively large.

[0010] It is an object of the present invention to provide a stain thatcreates a high contrast between the nucleus and the cytoplasm of a cell.It is a further object of the invention to stain the cytologicalmaterial such that the cytoplasm is relatively transparent to anautomated machine vision system, but visible to an observer.

[0011] It is another object of the invention to provide a stain withdual peak responsiveness when exposed to two distinct light wavelengthsand a system for verifying a specific stain was used based on the dualpeak responsiveness.

[0012] It is still a further object of the present invention to providea method of cytological analysis in which the cells are multicolored andthe nuclear portion is readily distinguishable from the cytoplasmicportion, both with automated imaging equipment and with human visionanalysis.

[0013] It is yet another object to provide a method of cytologicalanalysis in which the characteristics of the stained cells can beaccurately determined with both manual and automated analysisprocedures.

[0014] An additional object of the present invention is to provide asystem and method for illuminating cytological samples, wherein theillumination is supplied by two different light sources with twodifferent wavelengths. One light source verifies the stain used and theother light source facilitates analysis of the cytological sample.

SUMMARY OF THE INVENTION

[0015] Generally, the invention addresses the problems outlined above bymeans of unique stains, methods of staining cytological material, andillumination and imaging systems. Other techniques for addressing someof the drawbacks associated with the traditional Papanicaloau stains aredisclosed in U.S. Pat. No. 5,168,066, assigned to the same assignee asthe instant application, the disclosure of which is hereby incorporatedby reference in its entirety.

[0016] In one aspect, the invention relates to cytological stainingsolutions. The solutions include methanol, phenol or phenol derivatives,and thionin. In some embodiments, an acid may be present. The acid canbe almost any acid traditionally used to adjust the pH of a solution.For example, acetic acid, nitric acid, hydrochloric acid, phosphoricacid, formic acid, sulfuric acid, or citric acid could be used. Invarious embodiments, the methanol used can be various grades, the phenolor phenol derivative source can be loose crystals having an ACS grade ofat least about 95%, the thionin can be a certified dye powder, and theacid can be glacial acetic acid of various grades. The ACS grade is anindicator of the purity of the components. Components in accordance withequivalent grading systems are acceptable. In addition, the thionin canbe synthesized. In one embodiment, the staining solution has an acidicpH value, preferably about 5-7, and more preferably about 6.70+/−0.05.In one embodiment, the phenol or phenol derivative has a weight tovolume ratio of about 0.8% to about 1.2%, and preferably about 1.0%. Inanother embodiment, the thionin has a weight to volume ratio of about0.2% to about 0.5%, preferably about 0.3% to about 0.4%, and morepreferably about 0.345%. The weight to volume ratio, expressed as wt/v,is a measure of the weight of any one component as a percentage of thevolume of the entire solution.

[0017] In another aspect, the invention relates to methods of producingstaining solutions. The methods include the steps of mixing methanol,phenol or phenol derivatives, and thionin, stirring the mixture,filtering the mixture, and adding an acid to adjust the pH value of themixture to about 6.7. The acid is preferably added slowly whilestirring. As discussed above, the acid can be almost any acidtraditionally used to adjust the pH of a solution. In one embodiment,the mixture includes an equivalent ratio of about one liter of methanol,about 10 grams of phenol or phenol derivative, and about 3.45 grams oforganic or synthetic thionin. The mixture may be filtered using a filterwith about 1-20 micron particle retention. The mixture may be stirredfor at least about 1 hour.

[0018] In still another aspect, the invention relates to a cytologicalcounterstaining solution. Counterstaining is where the cells are stainedwith one or more dyes that are primarily taken up by the cytoplasm. Thesolution includes a reagent alcohol, eosin Y, thionin, and light or fastgreen. In various embodiments, the reagent alcohol can be 200 proof, theeosin Y, the thionin, and the light or fast green source can becertified dye powders, and an acid may be present, such as glacialacetic acid, ACS grade 99%. The thionin can be organic or synthetic. Inone embodiment, the staining solution has an acidic pH value, preferablyabout 5-6 and more preferably about 5.50+/−0.05. In another embodiment,the reagent alcohol consists of about 90% ethanol, 5% isoproponal, andabout 5% methanol. In one embodiment, the eosin Y has a wt/v ratio ofabout 0.05% to about 0.1%, preferably about 0.067% to about 0.08%, andmore preferably about 0.0721%. In another embodiment, the thionin haswt/v ratio of about 0.01% to about 0.03%, preferably about 0.015% toabout 0.025%, and more preferably about 0.0171%. In another embodiment,the light or fast green has a wt/v ratio of about 0.015% to about 0.03%and preferably about 0.0231%.

[0019] In yet another aspect, the invention relates to a method ofproducing a counterstaining solution. The method includes the steps ofmixing a reagent alcohol, eosin Y, thionin, and light or fast green,stirring the mixture, filtering the mixture, and adding an acid toadjust the pH value of the mixture to about 5.5. As discussed above, theacid can be almost any acid traditionally used to adjust the pH of asolution. In one embodiment, the mixture includes an equivalent amountof about one liter of reagent alcohol, about 0.721 grams of eosin Y,about 0.171 grams of organic or synthetic thionin, and about 0.231 gramsof light or fast green. In other embodiments, the mixture is filteredusing a filter with about 1-20 micron particle retention. The mixturemay be stirred for at least about 1 hour.

[0020] In still yet another aspect, the invention relates to a method ofstaining cytological material with thionin-phenol derivative solutions.The thionin-phenol derivative solutions bind preferentially to nuclearcytological material relative to cytoplasmic cytological material. Thesolutions can include methanol, phenol derivatives, and thionin, and canbe about a 0.3% thionin solution. The thionin can be organic orsynthetic. In one embodiment, the cytological material is dipped into asalt bath prior to staining. The salt bath can be about a 10% saltsolution. In another embodiment, the method includes the step ofcounterstaining the cytological material after staining with thethionin-phenol derivative solution.

[0021] Various embodiments of this aspect of the invention can includethe following features. The cytological material can be rinsed or dippedin an alcohol bath prior to counterstaining in a counterstain solution,which includes a reagent alcohol, eosin Y, thionin, and light or fastgreen. The thionin can be organic or synthetic. The counterstainingsolution binds preferentially to cytoplasmic cytological materialrelative to nuclear cytological material. The counterstaining solutioncan include at least one of the components of the thionin-phenolderivative stain, thionin in one embodiment, such that the thioninsubstantially replenishes the thionin depleted during the rinsing. Atleast one of the thionin-phenol derivative solution and thecounterstaining solution discernibly stain the cytological material inthe visible light range, or both the thionin-phenol derivative solutionand the counterstaining solution discernibly stain the cytologicalmaterial in the visible light range. Additionally, the cytologicalmaterial can be rinsed or dipped into an alcohol bath aftercounterstaining.

[0022] An additional aspect of the invention relates to counterstainingpreviously stained material, wherein the counterstain includes acomponent from the previous stain. The process of counterstaining thepreviously stained material replenishes any loss of the previous staincomponent that may have occurred subsequent to the initial staining, forexample, through rinsing the material after staining. In one embodimentof this method, the previously stained material is rinsed prior tocounterstaining. The rinse can be an alcohol bath.

[0023] Another aspect of the invention relates to an optical instrumentlighting system that includes a first light source and a second lightsource. The first light source has a first wavelength, which may bebetween about 690 nm and about 750 nm. The first light source is used toverify a stain used on a cytological sample. The second light source hasa second wavelength different than the first wavelength, and may bebetween about 500 nm and about 600 run. The second light source is usedto illuminate the cytological sample for viewing. In variousembodiments, the first light source can be a red light emitting diode(LED) and the second light source can be a green LED or an array of upto eight or more green LEDs. The light source can operate on lowvoltage, such as 5 volts DC. LEDs are bright, stable, and available in avery wide range of illumination wavelengths. Furthermore, LEDsefficiently produce a narrow band of illumination (typically 15 nm),eliminating the need for narrow band filters and allowing all energy tobe put into the desired illumination wavelength. Conventionalillumination, such as tungsten-halogen bulbs, put out a lot of wastedlight and heat when narrow band illumination is desired. LEDs generatesignificantly less heat and require substantially less power thanconventional imaging light sources. In fact, standard light sources areoften insufficient to obtain the necessary shutter times. In addition,LEDs are relatively tiny.

[0024] A further aspect of the invention relates to an LED array for usewith a system for imaging a cytological sample. The LED array includes ared LED for verifying the stained sample was stained with apredetermined stain and a green LED for illuminating the cytologicalsample for imaging. The array can include up to eight or more green LEDsand can operate on low voltage, such as 5 volts DC.

[0025] A still further aspect of the invention relates to a system forimaging a cytological sample including nuclear material and cytoplasmicmaterial. The system includes an optical instrument and first and secondlight sources. The first light source having a first wavelength forverifying that a specific stain was used on the cytological sample,wherein the specific stain will permit transmission of light at awavelength of about 720 nm. The second light source has a secondwavelength for illuminating the cytological sample for imaging, whereinthe stained nucleus of the sample will permit transmission of light at awavelength of about 570 nm and the cytoplasm will be essentiallyinvisible to the system. In one embodiment, the stain is athionin-phenol derivative solution, preferably about a 0.3% thioninsolution. In another embodiment, the cytoplasm is visible to anobserver. In various embodiments, the first light source can be a redLED and the second light source can be a green LED or an array of up toeight or more green LEDs. The light sources can operate on low voltage,such as 5 volts DC.

[0026] In yet another aspect, the invention relates to a method ofimaging a cell. The method includes the steps of staining the nuclearmaterial of the cell, staining the cytoplasmic material of the cell,illuminating the cell with a first light source having a firstwavelength for verifying that a specific stain was used, andilluminating the cell with a second light source having a secondwavelength for imaging the nuclear material.

[0027] Additional embodiments according to the foregoing aspect of theinvention may include the following features. The first wavelength isbetween about 690 nm and about 750 nm, and the second wavelength isbetween about 500 nm and about 600 nm. The first light source can be ared LED and the second light source can be a green LED or an array of upto eight or more green LEDs. The light sources can operate on lowvoltage, such as 5 volts DC. The stain used produces a high contrastbetween the nuclear material and the cytoplasmic material, such that thecytoplasmic material is relatively transparent, or invisible, to animaging system viewing the cell, but visible to an observer. Inparticular the nuclear material of the cell may be stained with athionin-phenol derivative solution, preferably about a 0.3% thioninsolution.

[0028] In still yet another aspect, the invention relates to a methodfor verifying that a specific stain was used on a cytological sample.The method includes the steps of staining the cytological sample with astain having two spectral peaks, at first and second wavelengths, andilluminating the sample with first and second light sources. The firstlight source has a first wavelength, wherein the specific stain willpermit transmission of light at a wavelength of about 720 nm. The secondlight source has a second wavelength, wherein the specific stain willpermit transmission of light at a wavelength of about 570 nm.

[0029] Further embodiments according to the foregoing aspect of theinvention may include the following features. The first wavelength isbetween about 690 nm and about 750 mn, and the second wavelength isbetween about 500 nm and about 600 nm. The first light source can be ared LED and the second light source can be a green LED or an array of upto eight or more green LEDs. The light sources can operate on 5 voltsDC. The cytological sample may be stained with a thionin-phenolderivative solution, preferably about a 0.3% thionin solution.

[0030] In a further aspect, the invention relates to a method of imaginga specimen. The method includes the steps of loading a specimen stainedwith a thionin-phenol solution into an optical instrument, illuminatingthe specimen, and imaging the specimen. The method can also include thestep of processing the image of the specimen. In one embodiment, thethionin-phenol solution includes a phenol derivative.

[0031] The invention also relates to a system for imaging a specimenstained with a thionin-phenol derivative solution. The system includesan optical instrument, at least one light source, a processor, and thestained specimen.

[0032] These and other objects, along with advantages and features ofthe present invention herein disclosed, will become apparent throughreference to the following description of embodiments of the invention,the accompanying drawings, and the claims. Furthermore, it is to beunderstood that the features of the various embodiments described hereinare not mutually exclusive and can exist in various combinations andpermutations.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In the drawings, like reference characters generally refer to thesame parts throughout the different views. Also, the drawings are notnecessarily to scale, emphasis instead generally being placed uponillustrating the principles of the invention. In the followingdescription, various embodiments of the present invention are describedwith reference to the following drawings, in which:

[0034]FIG. 1 is a chart depicting the characteristics and specificationsof a thionin dye;

[0035]FIG. 2 is a flow chart of one method of producing a thionin-phenolderivative stain;

[0036]FIG. 3 is a chart depicting the characteristics and specificationsof an eosin Y dye;

[0037]FIG. 4 is a chart depicting the characteristics and specificationsof a light green SF yellowish dye;

[0038]FIG. 5 is a flow chart of one method of producing a counterstain;

[0039]FIG. 6 is a flow chart of one method of staining a cytologicalmaterial;

[0040]FIG. 7 is a flow chart of another method of staining a cytologicalmaterial;

[0041]FIG. 8 is a flow chart of another method of staining a cytologicalmaterial;

[0042]FIG. 9 is a flow chart of another method of staining a cytologicalmaterial;

[0043]FIG. 10 is a schematic of an LED Koehler illumination system;

[0044]FIG. 11 is a schematic of an LED fiber illumination system;

[0045]FIG. 12 is a schematic of an optics system to couple two LEDs to afiber optic cable;

[0046]FIG. 13A is a schematic side view of an LED multichip module;

[0047]FIG. 13B is a schematic top view of an LED multichip module;

[0048]FIG. 14A is a schematic representation of an LED array;

[0049]FIG. 14B is a schematic representation of an alternative LEDarray;

[0050]FIG. 14C is a schematic representation of another alternative LEDarray;

[0051]FIG. 15 is a graph depicting an example of the absorption spectraof the individual components of the stain in solution;

[0052]FIG. 16 is a graph depicting another example of the absorptionspectra of the individual components of the stain in solution;

[0053]FIG. 17 is a graph depicting yet another example of the absorptionspectra of the individual components of the stain in solution;

[0054]FIG. 18 is a schematic representation of cytological material; and

[0055]FIG. 19 is a schematic representation of an imaging system andcooperating components in a laboratory setting.

DETAILED DESCRIPTION

[0056] Embodiments of the present invention are described below. It is,however, expressly noted that the present invention is not limited tothese embodiments, but rather the intention is that modifications thatare apparent to the person skilled in the art and equivalents thereofare also included.

[0057] One embodiment of the stain is a solution that includes methanol,phenol or phenol derivative, and thionin. The stain may also include anacid for adjusting the pH of the stain solution. The purity of themethanol (methyl alcohol) component can be various grades. The phenolcomponent is typically supplied as loose crystals having an ACS gradepurity of at least about 95%; however, the phenol may be in liquid form.The phenol is a chaotrophic agent available through Aldrich ChemicalCompany, Inc. of Milwaukee, Wis.; however, an equivalent may besubstituted. Phenol derivatives include: phenol ethers, such asphenoxyethanol, phenoxypropanol, phenoxyacetone, and phenylbutanol;phenol amines, such as phenylbutylamine; alkylphenols, such aspolymethylphenols, ethylphenols, isopropylphenols, sec-butylphenols,tert-butylphenols, tert-pentylphenols, cycloalkylphenols,aralkylphenols, alkenylphenols, and indanols; hydroxy benzene compounds;bisphenols; and halogenated phenolic compounds. In one embodiment, thephenol derivative is 2-phenoxyethanol, which may be obtained by treatingphenol with ethylene oxide in an alkaline solution. The thionincomponent is supplied as a certified dye powder, specifically aBSC-certified, metachromatic, cationic thiazine dye. FIG. 1 depicts thecharacteristics of one embodiment of the thionin dye used in the presentinvention as available from Aldrich Chemical Company or SIGMA of St.Louis, Mo.; however, equivalents may be used. The acid for adjusting thepH of the stain can be any one of most common acids, for example, aceticacid, citric acid, nitric acid, hydrochloric acid, phosphoric acid,sulfuric acid, or formic acid.

[0058] An acceptable weight per volume (wt/v) ratio of thionin tosolution is about 0.2% to about 0.5% wt/v, preferably about 0.3% toabout 0.4% wt/v, and more preferably about 0.345% wt/v. The ratio forthe phenol or phenol derivative is about 0.8% to about 1.2% wt/v andpreferably about 1.0% wt/v.

[0059] One method of producing the stain is depicted in FIG. 2. Mixing,step 5, entails adding the phenol or phenol derivative and thionin dyeto the methanol. Next, the mixture is stirred, step 10, optionallyvigorously for up to 15 hours . The mixture is then filtered, step 15.The mixture may be gravity filtered through a filter with about 1-20micron particle retention and preferably about 2 micron. One example isno. 4 filter paper as available from Whatmann. Finally, the pH level ofthe mixture is adjusted, step 20, by adding acid to the solution. Themixture may be stirred continuously while adding the acid. The pH isthen adjusted to about 5-7 and preferably about 6.7+/−0.05.Alternatively, one could measure the conductivity of the solution, step25, by any means known to confirm the correct composition of thesolution.

[0060] One embodiment of the counterstain, or second stain, includesreagent alcohol, eosin Y, thionin, and light or fast green. An acid foradjusting the pH of the counterstain solution may also be present. Thereagent alcohol is 200 proof and may include about 90% ethanol, 5%isopropanol, and 5% methanol. The eosin Y, thionin, and light or fastgreen are certified dye powders. The eosin Y is a fluorescent (yellow),red xanthene dye made by 2′, 4′, 5′, 7′-tetrabrominating fluorescein.Eosin Y is certified by BSC for use as a counterstain againsthematoxylin. FIG. 3 depicts the characteristics of one embodiment of theeosin Y dye used in the present invention, as available from AldrichChemical Company or SIGMA; however, equivalents may be used. The thioninis the same composition as used in the thionin-phenol derivativesolution. The light or fast green is a bluish-green, anionictriphenylmethane dye that is very soluble in water and slightly solublein ethanol. Light or fast green is BSC-certified for use as acytological counterstain. FIG. 4 depicts the characteristics of oneembodiment of a light green SF yellowish dye used in the presentinvention, as available from Aldrich Chemical Company or SIGMA; however,equivalents may be used. The acid for adjusting the pH of the stain canbe any one of most common acids, for example, acetic acid, citric acid,nitric acid, hydrochloric acid, sulfuric acid, phosphoric acid, orformic acid.

[0061] The ratio of eosin Y to solution for the counterstain is about0.05% to about 0.1% wt/v, preferably about 0.06% to 0.08% wt/v, and morepreferably about 0.0721% wt/v. The ratio for the thionin is about 0.01%to about 0.03% wt/v, preferably about 0.015% to 0.025% wt/v, and morepreferably about 0.0171% wt/v. The ratio for the light or fast green isabout 0.015% to about 0.03% wt/v and preferably about 0.0231% wt/v.

[0062] One method of producing the counterstain is depicted in FIG. 5.Mixing, step 30, includes adding the eosin Y, thionin, and light or fastgreen dyes to the reagent alcohol. Next, the mixture is stirred, step35, optionally vigorously, and preferably for up to 15 hours . Themixture is then filtered, step 40. The mixture may be gravity filteredthrough a filter with about 1-20 micron particle retention andpreferably about 2 micron. One example is no. 4 filter paper asavailable from Whatmann. Finally, the pH level of the mixture isadjusted, step 45, by adding acid to the solution. The mixture may bestirred continuously while adding the acid. The pH is then adjusted toabout 5 to 6 and preferably about 5.5+/−0.05. Alternatively, one couldmeasure the conductivity of the solution, step 25, by any means known toconfirm the correct composition of the solution.

[0063] A method of staining cytological material according to theinvention provides improved contrast of the nucleus relative to thecytoplasm over conventional staining methods. The method producesmulticolored cells suitable for manual analysis, and is also highlyeffective in automated analysis systems. The method entails the steps ofstaining the cytological material with a thionin-phenol derivativestain, counterstaining, illuminating the stained material, and imagingthe stained cytological material.

[0064] In one embodiment, the cells are stained by the method shown inFIG. 6. The cytological specimen is dipped or rinsed in a salt bath,step 55, of about a 10% salt solution. The solution is about 50%alcohol; wherein, the alcohol is 200 proof and consists of about 90%ethanol, about 5% isopropanol, and about 5% methanol. Step 60 entailsstaining the cells with a thionin-phenol stain. The stain may be about a0.3% thionin solution. Step 65 entails counterstaining the cells with asecond stain. The second stain may include thionin.

[0065] In another embodiment, the cells are stained by the method shownin FIG. 7. The cytological specimen is dipped or rinsed in a salt bath,step 55. Next the cells are dipped or rinsed in alcohol, step 70. Step60 entails staining the cells with a thionin-phenol derivative stain,and step 75 entails dipping or rinsing the cells in alcohol. Lastly,step 65 counter stains the cells with a second stain.

[0066] According to one practice of the invention, the cells are stainedby the general method shown in FIG. 8. The cells are fixed on the slide,step 80, rinsed in alcohol and/or water baths, step 85, rinsed in a saltbath, step 55, rinsed in alcohol, step 70, and stained with athionin-phenol derivative stain solution, step 60. The stained cells arerinsed in an alcohol bath, step 75, and counterstained, step 65. Aftercounterstaining, the stained cells are rinsed in alcohol, step 90, andrinsed in xylene or other commercially available xylene substitutes,step 95.

[0067] As shown in FIG. 9, the cells are first fixed on a slide usingalcohol and wax, or other methods known in the art, step 80. Then, inpreparation for staining, the slide is dipped ten to twenty times ineach of two baths, where one is a high percentage, low molecular weightalcohol, preferably ethanol, and the other is an alcohol bath or a waterbath, step 86. Next, the cells are dipped in a salt bath for about fiveto about fifteen minutes, generally for about ten minutes, step 55,after which the cells are rinsed in an alcohol bath, step 70. The cellsare then stained in a thionin-phenol derivative stain solution, step 60,for a time sufficient to incorporate the thionin dye in the nuclei,which may range from three minutes to about thirty minutes, but istypically about ten minutes. Those skilled in the art will recognizethat the staining time can be determined by taking several factors intoaccount, including the desired intensity of the stain appropriate forthe cell type and the viewing system used. Automated imaging systemsgenerally require darker staining than human visual evaluation, andcertain types of cells stain faster than others. Also, the amount ofthionin in the stain can affect the staining time. Lower concentrationsof thionin will generally require longer staining times. A thionin stainuseful for most cells is slightly acidic, typically having a pH of about6.7.

[0068] After rinsing by dipping ten to twenty times in each of two tothree high percentage, low molecular weight alcohol baths, followed by athird bath lasting approximately five minutes in a high percentage, lowmolecular weight alcohol, step 76, the cells are counterstained, step65, for a time sufficient to incorporate the dye in the cytoplasm andnuclei, which may range from about two minutes to about thirty minutes,but is generally about four minutes. Those skilled in the art willrecognize that the staining time may vary, as discussed hereinabove. Thecounterstain also includes thionin in an amount sufficient to replenishthe thionin that may have been depleted during the dipping and rinsingsteps, and is selected to absorb light at a different wavelength fromthe thionin-stained nuclear material. After staining andcounterstaining, the slide is rinsed by dipping in two to four more highpercentage, low molecular weight alcohol baths, step 91, and two or morexylene rinses or other commercially available xylene substitutes, step96. The cells are now ready to be analyzed.

[0069] When viewed under visible light, the nuclei of the cells aretransparent to opaque and stained a deep blue. The cytoplasm istransparent and is multicolored, with the specific color patterndepending on the counterstain used. When cells are stained in thismanner, the color pattern is familiar to cytologists, so analysis canreadily be carried out by manual, i.e., human vision. The method has theadded advantage in that, when viewed by an imaging system, each nucleusis opaque and the cytoplasm is nearly invisible, resulting in very highcontrast between nuclear and cytoplasmic material and thereby providinghigh resolution. With the cytoplasm nearly invisible, overlapping cellswill not be confused with nuclei, and an accurate cell count can beachieved readily, manually or by computer.

[0070] Referring now to FIGS. 10 and 11, one purpose of an opticalinstrument lighting system in accordance with the present invention isto provide high intensity narrow band light for imaging a cytologicalspecimen on a microscope slide 150. This purpose is implemented usinghigh brightness LEDs in either a single discrete LED, combination ofsingle discrete LEDs, or custom multi-chip LED module configuration 152.LEDs generate a small amount of heat, which restricts the amount ofcurrent at which they can be driven before output wavelengths shift,consequently, restricting the amount of light that these devices emit.To increase the light output from the device, the LEDs may be drivenwith a pulse circuit 160 that is synchronized with a camera 156 of theimaging system 164 to deliver short, intense pulses of light to thecamera during the camera integration period.

[0071] The LED pulse is synchronized with the camera frame integrationby providing an external trigger that triggers both the camera/framegrabber 162 and the LED pulse driver 160, for example, using a squarewave generator 158. The camera 156 does not respond instantaneously tothe trigger. To compensate for this delay, the pulse drive circuit 160has a programmable delay that is used to synchronize the systems. Othersynchronization methods are possible, depending on the camera type andactual implementation.

[0072] LEDs inherently produce spatially nonuniform light output andsome machine vision imaging applications require reasonably uniformillumination of the sample. For situations where uniformity is an issue,two types of systems for generating spatially uniform illumination maybe used with LEDs, namely, Koehler and fiber optic systems. The LEDsused in either of these systems may be discrete LEDs packaged in closeproximity or multiple LED dies may be integrated on a single substrateto produce a more dense arrangement.

[0073] Koehler illumination (see FIG. 10) is a standard technique forproducing uniform illumination of a microscope slide 150 from thespatially nonuniform filament of an incandescent lamp used intraditional microscope illuminators. As determined by testing, thistechnique is equally effective at achieving uniformity when employedwith LED sources. In this embodiment of the LED illuminator, individualLEDs are packaged closely together and placed in the general position ofthe lamp filament in the traditional Koehler illuminator 154 andpositioned for Koehler illumination.

[0074] In another embodiment for achieving uniformity, multiple LEDs arecoupled into a large core (=500 to 600 μm) optical fiber 166 with lensesor other optical apparatus. See FIG. 11. The length of the fiber isselected so that the spatial nonuniformities of the LEDs are mixedtogether and a relatively uniform spatial output from the fiber 166 isachieved. In practice, the output of the fiber 166 is approximatelyGaussian in spatial profile. The fiber 166 may have to be displaced fromthe microscope slide 150, such that only the central, relatively flat,portion of the output is used.

[0075] Consider the case where the multi-chip LED module 152 containstwo LEDs. Two discrete LEDs having a full-angle divergence of 8 degreesare placed side-by-side such that both emitters fall within a 4 mmdiameter. The lenses collect the light and image both emitters onto the600 μm core of the fiber 166. See FIG. 12. The package diameter ofindividual LEDs is typically on the order of 3-5 mm. Thus, it may benecessary to remove some of the nonessential plastic packaging on astandard LED in order to get the two emitters side-by-side within 4 mm.

[0076] As an alternative to individual LEDs, multiple LED dies 168 maybe placed on a single substrate 170 See FIGS. 13A and 13B. Individuallenses 172 can be placed above each die 168 so that the radiation fromeach die 168 is collected into a narrow cone, rather than being spreadinto 2π steradians. Microlenses with diameters of about 1 mm arecommercially available. A hexagonal pattern of 1 mm lenses could packabout twelve LED die 168 onto a single substrate 170 with a diameter ofabout 4 mm. A substrate 170, such as one with high thermal conductivity,may be used to hold multiple LED dies 168. Conductive patterns on thesubstrate 170 are used to wire bond the dies 168 to the substrate 170for electrical connections. The dies 168 are potted in a polymer, forexample an epoxy, for protection from moisture and other elements. Theindividual microlenses 172 are placed on top to provide collection ofthe light into a narrow cone.

[0077] An extension of this concept consists of combining differentwavelength LEDs to produce a light source that emits multiple narrowwavelength bands. This technique provides a method to illuminate objectssuch as cytological specimens on microscope slides at multiplewavelengths simultaneously. Further, the output bands can be tailored tothe application by selection of appropriate LEDs. The energy from eachband can be tailored to the application by employing separate drivesystems, pulsed or continuous as required, for each wavelength.

[0078] In one embodiment, a cluster or array of seven LEDs replaces thestandard incandescent microscope illumination. The LEDs are oriented inthe configuration shown in FIG. 14A. Different spatial orientations maybe used to obtain two colors. One, shown in FIG. 14B, provides for twotriplets of LEDs where “X” represents one color LED and “O” representsanother. Two wavelengths are selected, one allowing optimal nucleardetection among red cytoplasm and the other allowing optimal nucleardetection among green cytoplasm. The second orientation as shown in FIG.14C allows for nine LEDs, with four illuminating one color and fiveilluminating the other color. In additional embodiments, the LED arraymay include essentially any combination of the two different color LEDs,such as one red LED and eight green LEDs. Choices of the color andnumber of LEDs depends on desired brightness, camera response at theselected wavelengths, and availability of LEDs with specific emissionangles, for example, 35 degree and 65 degree emission angle LEDs.Typical angles range from about 24 to about 72 degrees. The red LEDs areused for exposing the stained cytological material to a light sourcehaving a wavelength of about 690 nm to about 750 nm to determine that aspecific stain was used. The green LEDs having a wavelength of about 500nm to about 600 nm are used to illuminate the cytological material forimaging. Multiple green LEDs are desirable, because increased imagingresponse times can be achieved with the increased illumination.

[0079] FIGS. 15-17 depict various examples of the absorption spectra ofthe individual components of the stains in solution. Two peaks arereadily apparent from the graphs. One peak is associated with red lightand the other is associated with green light. It is generally desirableto image the cytological material at a wavelength at which green and redlight are minimally transmitted, i.e., the valley formed between the twospectral peaks, in this instance about 568 nm.

[0080]FIG. 18 is a schematic representation of cytological material asit may be seen on a typical slide 300. Each cell 305 consists of anucleus 310 and cytoplasm 315. A representative abnormal nucleus 320 isalso shown.

[0081]FIG. 19 is a schematic representation of an imaging system andrelated components in a laboratory setting. The overall system 200 mayinclude a system 210 for preparing slides 250 with cytological materialfor review, an imager/processor 220 for imaging the cytological materialand processing the pertinent data, a computer server 230 for storingand/or further processing the data, and a reviewing station 240 formanually reviewing the slides based on data collected by the imager 220.The slide 250 may be stained with a thionin-phenol or thionin-phenolderivative solution, as described hereinabove.

[0082] Having described certain preferred and exemplary embodiments ofthe invention, it will be apparent to those of ordinary skill in the artthat other embodiments incorporating the concepts disclosed herein canbe used without departing from the spirit and the scope of theinvention. The described embodiments are to be considered in allrespects only as illustrative and not limiting. Therefore, it isintended that the scope of the present invention be only limited by thefollowing claims.

What is claimed is:
 1. A cytological staining solution, comprising: methanol; a phenol derivative; and thionin.
 2. The staining solution of claim 1, wherein the methanol is various grades.
 3. The staining solution of claim 1, wherein the phenol derivative is selected from the group consisting of phenol ethers, phenol amines, akylphenols, hydroxy benzene compounds, bisphenols, and halogenated phenolic compounds.
 4. The staining solution of claim 1, wherein the phenol derivative is phenoxyethanol.
 5. The staining solution of claim 1, wherein the thionin comprises a certified dye powder.
 6. The staining solution of claim 1 further comprising an acid, wherein the acid is selected from the group consisting of acetic acid, phosphoric acid, nitric acid, formic acid, citric acid, sulfuric acid, and hydrochloric acid.
 7. The staining solution of claim 1, wherein the acid is glacial acetic acid.
 8. The staining solution of claim 1, wherein pH of the staining solution is acidic.
 9. The staining solution of claim 1, wherein pH of the staining solution is between about 5 to about
 7. 10. The staining solution of claim 1, wherein pH of the staining solution is about 6.7+/−0.05.
 11. The staining solution of claim 1, wherein the phenol derivative has a weight to volume ratio of about 0.8% to about 1.2%.
 12. The staining solution of claim 1, wherein the phenol derivative has a weight to volume ratio of about 1%.
 13. The staining solution of claim 1, wherein the thionin is synthesized.
 14. The staining solution of claim 1, wherein the thionin has a weight to volume ratio of about 0.2% to about 0.4%.
 15. The staining solution of claim 1, wherein the thionin has a weight to volume ratio of about 0.3%.
 16. The staining solution of claim 1, wherein the thionin has a weight to volume ratio of about 0.345%.
 17. A method of producing a cytological staining solution, the method comprising the steps of: mixing methanol, a phenol derivative, and thionin; stirring the mixture; filtering the mixture; and adjusting pH of the mixture to a value of about 6.7 by adding an acid thereto to produce the staining solution.
 18. The staining solution of claim 17, wherein the phenol derivative is selected from the group consisting of phenol ethers, phenol amines, akylphenols, hydroxy benzene compounds, bisphenols, and halogenated phenolic compounds.
 19. The staining solution of claim 17, wherein the phenol derivative is phenoxyethanol.
 20. The method of claim 17, wherein the acid is chosen from the group consisting of acetic acid, phosphoric acid, nitric acid, formic acid, citric acid, sulfuric acid, and hydrochloric acid.
 21. The method of claim 17, wherein the acid is glacial acetic acid.
 22. The method of claim 17, wherein the mixture is mixed for at least about 1 hour.
 23. The method of claim 17, further comprising the step of mixing the mixture while adding the acid.
 24. The method of claim 17, wherein the mixing step comprises mixing an equivalent ratio of about one liter of methanol, about 10 grams of phenol derivative, and about 3.45 grams of thionin.
 25. The method of claim 17, wherein the filtering step utilizes a filter with about 1-20 micron particle retention.
 26. A method of staining cytological material, the method comprising staining the cytological material with a thionin-phenol derivative solution.
 27. The method of claim 26, further comprising the step of rinsing the cytological material in a salt bath prior to staining.
 28. The method of claim 26, further comprising the step of counterstaining the cytological material with a second stain.
 29. The method of claim 27, wherein the salt bath is about a 10% salt solution.
 30. The method of claim 26, wherein the thionin-phenol derivative solution is about a 0.3% thionin solution.
 31. The method of claim 26, wherein the thionin-phenol derivative solution comprises methanol, a phenol derivative, and thionin.
 32. The method of claim 26, wherein the thionin is synthesized.
 33. The method of claim 28, wherein the second stain comprises reagent alcohol, eosin Y, thionin, and light or fast green.
 34. The method of claim 26, wherein the thionin-phenol derivative solution binds preferentially to nuclear cytological material relative to cytoplasmic cytological material.
 35. The method of claim 28, wherein the second stain binds preferentially to cytoplasmic cytology material relative to nuclear cytological material.
 36. The method of claim 28, wherein at least one of the thionin-phenol derivative solution and the second stain discernibly stain the cytological material in a visible light range.
 37. The method of claim 28, wherein both the thionin-phenol derivative solution and the second stain discernibly stain the cytological material in a visible light range.
 38. The method of claim 26, further comprising the step of rinsing the cytological material after staining the cytological material.
 39. The method of claim 28, wherein the second stain contains thionin to retard thionin depletion during the rinsing step.
 40. The method of claim 28, wherein the cytological material is rinsed in an alcohol bath.
 41. A method of imaging a specimen, the method comprising the steps of: loading a specimen stained with a thionin-phenol solution into an optical instrument; illuminating the specimen; and imaging the specimen.
 42. The method of claim 41, further comprising the step of processing the image of the specimen.
 43. The method of claim 41, wherein the thionin-phenol solution comprises a phenol derivative.
 44. A system for imaging a specimen stained with a thionin-phenol derivative solution, the system comprising: an optical instrument; at least one light source; a processor; and the stained specimen.
 45. A cytological counterstaining solution, comprising: reagent alcohol; eosin Y; thionin; and light or fast green.
 46. The counterstaining solution of claim 45, wherein the light or fast green comprises a certified dye powder.
 47. The counterstaining solution of claim 45, wherein the light or fast green has a weight to volume ratio of about 0.015% to about 0.03% for the light green and about 0.005% to about 0.01% for the fast green.
 48. A method of producing a cytological counterstaining solution, the method comprising the steps of: mixing reagent alcohol, eosin Y, thionin, and light or fast green; stirring the mixture; filtering the mixture; and adjusting pH of the mixture to a value of about 5.5 by adding an acid thereto to produce the counterstaining solution.
 49. The method of claim 48, wherein the mixing step comprises mixing an equivalent ratio of about one liter of reagent alcohol, about 0.721 grams of eosin Y, about 0.171 grams of thionin, and about 0.231 grams of light green or about 0.065 grams of fast green. 